Characterization of immobilized enzymes in polyurethane foams in a dynamic bed reactor

beta-D-Galactosidase (E 3.2.1.23) from Aspergillus oryzae was immobilized with polyurethane foam (PUF). Among several immobilization methods attempted in this work, the immobilized enzyme preparation by in-situ co-polymerization between enzyme and prepolymer HYPOL 3000 showed the highest activity. The intrinsic kinetics of PUF-immobilized enzyme was determined in a dynamic bed reactor, used to increase transport rates. The immobilization mechanism in PUF was studied by measurements of immobilized enzyme kinetics and by using scanning electron microscopy combined with immuno-gold labeling techniques. The results showed that immobilization was predominantly by covalent bonding between primary amino groups of beta-D-galactosidase and isocyanate groups of the prepolymers. Entrapment in the PUF micropores assisted the immobilization of enzymes, and adsorption on the surface of macropores was not important for immobilization. The bicinchoninic acid method was applied for the determination of PUF loading capacity and specific enzyme activity and used to determine enzyme deactivation during immobilization.